Dynamics of MAP kinase cascade in immune response

免疫反应中 MAP 激酶级联的动态

基本信息

批准号：
14572036
负责人：
SUZUKI Ryo
金额：
$ 2.24万
依托单位：
Nagoya City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14572036/
关键词：
MAP kinase cascade Mast Cell Immune Response Fluorescent Protein Confocal Laser Scanning Microscopy Membrane Translocation Nuclear shuttling Nuclear Export Signal 蛍光蛋白質GFP

项目摘要

The MAP kinase cascade is essential for signaling of various extracellular stimuli to the nucleus. Stimulation of T cells, B cells, and mast cells through aggregation of antigen receptors and other stimulants results in activation of MAP kinase. In this study, we focused on the dynamics of MAP kinase (MAPK) cascade protein in mast cells using yellow fluorescent protein (YFP)-tagged MAPK (ERK2) and YFP-tagged MAPKKK (Raf-1). Here, we have shown many aspects of MAP kinase signaling in immune response.1.In wild type mast cells, the membrane translocation of MAPKKK (Raf-1) reached 〜3 min after antigen stimulation, and thereafter the nuclear import of MAPK (ERK2) reached the maximum at 5〜7 min Raf-1 did not co-localize with lipid microdomain.2.In the presence of leptomycin B (inhibitor of nuclear export), the export of ERK2 became more slowly though ERK2 was imported to the nucleus as like as in non-treated cells.3.We have studied the effects of FcγRIIB on the FcεRI-mediated nuclear shuttling of MAP kinase in immune cells. The co-clustering of FcεRI and FcγRIIB increased the [Ca-<2+>]_i and induced the nuclear import of ERK2. However, the calcium increase was transient and ERK2 was rapidly exported to the cytoplasm.4.We have studied the effects of two tyrosine phosphatases, HePTP (a tyrosine-specific phosphatase) and VHR (a dual specificity protein-tyrosine phosphatase), on the FcεRI-mediated nuclear shuttling of ERK2 in the mast cells. In HePTP-overexpressing cells, the maximal amount of ERK2 accumulated in the nucleus was much less than that in wild type cells though the nuclear import of ERK2 reached the maximum in the similar time-course. In VHR-overexpressing cells, the amount of imported ERK2 decreased more rapidly though ERK2 was imported to the nucleus as like as in wild type cells.Thus we succeeded to show the molecular mechanism of MAP kinase cascade protein in immune response.

MAP 激酶级联对于将各种细胞外刺激信号传递至细胞核至关重要。通过抗原受体和其他刺激物的聚集刺激 MAP 激酶，从而激活 T 细胞、B 细胞和肥大细胞。使用黄色荧光蛋白 (YFP) 标记的 MAPK (ERK2) 和 YFP 标记的 MAPKKK (Raf-1) 来研究肥大细胞中 MAP 激酶 (MAPK) 级联蛋白的动态。已经显示了免疫反应中 MAP 激酶信号传导的许多方面。1.在野生型肥大细胞中，MAPKKK (Raf-1) 的膜易位在抗原刺激后达到约 3 分钟，此后 MAPK (ERK2) 的核输入达到Raf-1在5〜7分钟时达到最大值 Raf-1没有与脂质微结构域共定位。2.在leptomycin B（核输出抑制剂）存在下，ERK2的输出变得更慢，尽管ERK2与未处理的细胞一样被输入细胞核。3.我们研究了FcγRIIB对免疫细胞中FcεRI介导的MAP激酶核穿梭的影响。FcεRI和FcγRIIB的共聚增加了[Ca-]。 <2+>]_i并诱导ERK2的核输入，但是钙的增加是短暂的并且ERK2迅速输出到细胞质。4.我们研究了两种的影响。酪氨酸磷酸酶、HePTP（一种酪氨酸特异性磷酸酶）和 VHR（一种双特异性蛋白酪氨酸磷酸酶）对肥大细胞中 FcεRI 介导的 ERK2 核穿梭的影响。尽管ERK2的核输入在野生型细胞中达到最大，但细胞核比野生型细胞少得多。在VHR过表达的细胞中，尽管ERK2像野生型细胞一样被导入细胞核，但导入的ERK2的量下降得更快。因此，我们成功地展示了MAP激酶级联蛋白在免疫反应中的分子机制。。